Microfluidic devices may be used in a variety of assays where the capture or modification of target analytes (or substrates) or localized chemical environment affecting target molecules is desired. Generally, microfluidic devices suitable for capturing target analytes may employ biomolecules, antibodies, or other affinity reagents including, but not limited to, DNA probes, RNA probes, aptamers, thioaptamers, antibody fragments, lectins, cell surface receptors, streptavidin and other receptors or ligands immobilized to a surface of the device or a feature within the device. In some devices used for modifying a localized chemical environment a local charge may be modified to facilitate electro-osmotic flow or define a local pH, for example. In other examples, a hydrophobicity or hydrophilicity (e.g., PEG grafting) of a surface may be altered or selected to chemically modify a localized environment.
Accordingly, antibodies may be immobilized to a surface (e.g. electrode, glass, or other two-dimensional surface) of a microfluidic device through an appropriate chemical reaction or treatment. Bulk fluid flow containing the target analyte may then be passed across the treated surface, and target analytes may diffuse down to bind with the antibodies. Subsequent steps are required to detect the bound antigen (e.g. ELISA).
Generally, a multi-step process is used to graft a biomolecule (among others, proteins, antibodies, enzymes, or DNA molecules) to a porous polymer monolith surface. A common approach is to first define a porous polymer monolith framework fabricated with a chemically reactive monomer in the precursor solution (for example glycidyl methacrylate). Biomolecules are subsequently grafted to the surfaces of the porous monolith framework. Biomolecules can be chemically grafted directly to the monolith (for example via amine groups of the biomolecule reacting with the epoxide functionality of a GMA-based monolith). However, intermediate modifications to the monolith framework have been preferred to achieve higher grafting efficiencies. For example, the epoxide functionality of GMA-based monoliths has been modified by i) aminolysis followed by activation with dialdehyde, ii) hydrolysis of epoxide groups followed by oxidation, or iii) hydrolysis of epoxides followed by activation with carbonyldiimidazole before final reaction of these modified functionalities with amine groups of the biomolecule.
Porous polymer monolith frameworks have also been modified or activated in desired regions via photolithography before grafting biomolecules. For example, in a two-step process, a solution containing the photoinitiator benzophenone is loaded into the monolith framework. Upon exposure to UV, the benzophenone molecule abstracts hydrogen from the polymer surface and becomes tethered to the surface only in the UV exposed region or regions. A second solution containing 4,4-dimetheyl-vinylazlactone is loaded into the monolith and tethered to the surface via reaction with the benzophenone. The covalently linked azlactone functionality of the 4,4-dimetheyl-vinylazlactone can react with amine groups present on many biomolecules. The net result is that biomolecule immobilization is isolated to the UV activated regions of the monolith that contain the surface grafted benzophenone. Single step processes have also been demonstrated where benzophenone and 4,4-dimetheyl-vinylazlactone are loaded into the monolith and polymerized simultaneously but may polymerize within the pores and clog the monolith.
In another process, the monolith precursor solution may include a photoiniferter species and solid salt particles. After polymerization of the monolith, a porous polymer network structure may be formed by dissolving the salt particles by introducing deionized water. The pores in the monolith are defined by the salt particle size and distribution. An example of this leaching process is described generally in Simms, et. al., “In situ fabrication of macroporous polymer networks within microfluidic devices by living radical photopolymerization and leaching,” Lab Chip, 2005, 5, 151-157, which article is incorporated by reference herein in its entirety for any purpose.